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Title Study on structure and function of sucrosephosphate synthase from sugarcane

Author(s) Sawitri, Widhi Dyah

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URL https://doi.org/10.18910/56056

DOI 10.18910/56056

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Study on structure and function of sucrose phosphate synthase from sugarcane

サトウキビ由来 sucrose phosphate synthaseの構造機能研究

A doctoral thesis

Widhi Dyah Sawitri

Departement of Biological Sciences, Graduate School of Science Laboratory of Supramolecular Crystallography, Institute for Protein

Research Osaka University

February 2016

Abbreviation

DTT; dithiothreitol

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EDTA; ethylenediaminetetraacetic acid

F6P; fructose-6-phosphate

GFP; green flouresence protein

G6P; glucose-6-phosphate

GTD; glucosyl-transferase domain

IPTG; isopropyl β-D-1-thiogalactopyranoside

LB; luria broth

PHD; phosphohydrolase domain

PMSF; phenylmethylsulfonyl fluoride

SDS-PAGE; sodium dodecyl sulfate poly-acrylamide gel electrophoresis

SPS; sucrose phosphate synthase

S6P; sucrose-6-phosphate

TEV; tobacco etch virus

UDP-G; uridine diphosphate glucose

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General Introduction

Sucrose biosynthesis

It has been discovered that sucrose is the most common sugar in plant life,

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including unicellular algae and cyanobacteria. Sucrose biosynthesis occupies a crucial

role in the functional biology of plant, whereas the function of sucrose in

microorganism is still unanswered. The recent biochemical and molecular studies of

sucrose biosynthesis in prokaryotic offer new insight into the origin of sucrose

metabolism. Sucrose is the major products of plant photosynthesis transported from

leaves to provide the rest of plant for growth, development, signal transduction, and

storage reserves for allocation carbon resources in some plants (Lunn, J. E., et al 2003b;

Salerno, G. L., et al 2003).

The enzymes are responsible for sucrose metabolism has been characterized

in sucrose biosynthesis related proteins (SBRPs) including sucrose-phosphate synthase

(SPS), sucrose synthase (SuS), and sucrose-phosphate phosphatase (SPP) (Cumino, A.,

et al 2002; Salerno, G. L., et al 2003). SPS plays a key role in the pathway of sucrose

synthesis, which catalyzes the formation of sucrose-6-phosphate (S6P) from uridine

diphosphate glucose (UDP-G) and fructose-6-phosphate (F6P) as substrates (Amir, J., et

al 1982; Leloir, L. F., et al 1954; Huber, S. C., et al 1996). S6P is further hydrolyzed to

sucrose by SPP. The hydrolysis of the intermediate by SPP leads to an essentially

irreversible pathway providing an efficient production of substrate. SuS involves a

reversible reaction in the sucrose synthesis and cleavage. However, plant SuS generally

has been associated with cleavage of sucrose and UDP to fructose and UDP-G. Another

enzyme has been linked with sucrose cleavage is invertase. Otherwise, invertase

catalyzes the irreversible cleavage sucrose to glucose and fructose (Fig. 1) (Koch, K., et

al 2004; Salerno, G. L., et al 2003; Winter, H., et al 2000).

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Fig. 1-1 Scheme of the sucrose biosynthesis

The structure of SBRPs in plant and cyanobacteria are suggested to be

multiple-domain proteins and it may have originated from primordial functional

domains of sucrose biosynthesis. The domainal protein of the most SBRPs is a

functional glucosyl-transferase domain (GTD) and phosphohydrolase domain (PHD)

(Fig. 1-2). SuS and SPS are sharing conserved residues with functional GTD and

cyanobacterial SPP defined a PHD of SBRPs. The characteristic of the structural

features of SBRPs is including the extension of N-terminal at GTD of SuS, N-terminal

at GTD of plant SPS, and C-terminal at PHD of plant SPS. All of the extensions have

been suggested to represent oligomeric proteins (Cumino, A., et al 2002; Salerno, G. L.,

et al 2003).

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Fig. 1-2 Structure analysis of SBRPs

The biochemical studies in less-complex organism, such as cyanobacteria,

proteobacteria, and lower plants, has recently increased our knowledge of new

perspective on plant sucrose metabolism and the structural studies of SBRPs will give

an important contribution to their catalytic and regulatory properties (Salerno, G. L., et

al 2003). However, the crystal structure of plant SPS which has pivotal role for

synthesis of sucrose is still unknown. Therefore, structural studies of plant SPS is

necessary to draw more extensive story of mechanism on sucrose metabolism.

Regulation of sucrose phosphate synthase (SPS) in higher plants

SPS catalyzes the transfer of glycosyl group of UDP-G and F6P as substrates

to S6P. It has been believed that SPS plays the physiological role of regulating

photosynthetic carbon flux into sucrose. The activity of SPS is regulated by diurnal

cycles of day/night (Ohsugi, R., et al 1987) and other environmental factors such as

osmotic stress (Quick, P., et al 1989) and cold temperature (Hurry, V. M., et al 1994).

Plant SPS is under complex regulation involving allosteric control by

metabolites, such as glucose-6-phospate (G6P) and inorganic phosphate (Pi) (Fig. 1-3).

An increase in G6P and a decrease in Pi, as might occur during dark to light transition

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and it would favor activation of SPS. Simultaneously, the catalytic activity would be

increased as the result of allosteric regulation (Huber, S. C., et al 1996).

Fig. 1-3 SPS regulation in higher plant

Plant SPSs have a molecular mass around 120 kDa and consists of three

domains. The central region contains glycosyltransferase domain responsible for the

catalytic function of SPS (Castleden, C. K., et al 2004). The C-terminal region

resembles sucrose phosphate phosphatase and the N-terminal region has no clear

similarity with any other proteins. It has been proved by biochemical and sequential

analysis that SPS has three different domains: the minimal catalytic SPS unit which

defines a functional GTD; GTD with PHD; and plant SPS which consists of N-terminal

regulatory region, GTD, and PHD (Fig. 1-2) (Salerno, G. L., et al 2003). The domains

arrangement would be responsive to different mechanism for controlling the

metabolism of sucrose. Notably, cyanobacteria SPSs (Cumino, A., et al 2002) and

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bacterial SPSs (Chua, T. K., et al 2008) which do not appear to have N-terminal region

are not activated by allosteric effectors.

The knowledge of plant SPS regulatory function still remains unclear. With

the aim of studying biochemical and structural basis of plant SPS, it would give an

important insight into the reaction mechanism of plant SPS as well as the bacterial

enzyme.

Characterization of SPS from sugarcane

Sucrose is the main storage reserve in sugarcane (Saccharum officinarum)

and the accumulation of sucrose has been dependent on enzymatic activity of sucrose

synthesis, such as SPS (Zhu, Y. J., et al 1997; Castleden, C. K., et al 2004). In some

plants, including sugarcane, SPS plays role not only as a regulator of photosynthetic

genes expression but also of non-photosynthetic genes. In the previous study, it was

identified two cDNAs encoding sugarcane SPS with different characterization in the

photosynthetic and non-photosynthetic tissues (Sugiharto, B., et al 1997).

Two clones encoding SPS were obtained from cDNA library of sugarcane

leaves. The comparison between deduced amino acid of sugarcane SPS, SoSPS1 and

SoSPS2, and SPSs from other species showed that SoSPS1 had the highest homology

about 95% identical to maize SPS and less but significant homology to spinach SPS

(56%), sugar beet SPS (56%), and potato SPS (55%). SoSPS2 had significant homology

to maize (50% identical), spinach (58%), sugar beet (57%), and potato (56%). The

corresponding sequences revealed that 49% identity between SoSPS1 and SoSPS2

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(Sugiharto, B., et al 1997).

It also has been reported that SoSPS1 and SoSPS2 are accumulated in

different organs and functional differentiation. SoSPS1 was differentially regulated in

response to light and predominantly detected in leaves, whereas SoSPS2 was not

affected by light and slightly expressed at similar level in leaves and roots (Sugiharto,

B., et al 1997).

Carbon partitioning is a critical process in distributing chemical energy

converted by plant through photosynthesis. As noted above, the regulation of SoSPS1

activity is representative for sucrose biosynthesis enzyme which is regulated in the

photosynthetic tissue. In this thesis, SoSPS1 has been used for understanding the plant

SPS regulation in sugarcane.

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Study 1.

Expression of SoSPS1 in E. coli

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Introduction

The crystal structure of SPS from the nonphotosynthetic bacterium

Halothermothrix orenii was reported and the structure revealed the mechanisms of

substrate binding and glycosyl transfer (Chua, T. K., et al 2008). This knowledge could

give an important insight into the reaction mechanism of plant SPS as well as the

bacterial enzyme. However, the structural basis for the regulatory function of plant SPS

still remains totally unknown. Unlike plant SPS, bacterial enzyme activity is not

allosterically regulated. Approximately 180 amino acids at N-terminal region and

C-terminal region of plant SPS are missing in H. orenii bactaria SPS (Chua, T. K., et al

2008).

It was previously cloned cDNAs from sugarcane leaves, SoSPS1 and SoSPS2

encoding two isoforms of SPS. SoSPS1 is abundantly expressed in sugarcane leaves and

is considered to be a representative of the isoenzyme responsible for photosynthetic

carbon allocation with regulatory function (Sugiharto, B., et al 1997). In this study,

cDNA of SoSPS1 was expressed in E. coli and with the aim of studying

structure-function relationship of the regulatory mechanism of the plant enzyme, the

recombinant plant SPS with quality and amount sufficient for biochemical analysis has

been prepared.

Material and methods

Construction of various expression plasmids of sugarcane SoSPS1 cDNA in E. coli

To allow the expression of full size enzyme encoded by sugarcane SoSPS1

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(gene accession No. AB001337 in GenBank) in E. coli, NcoI/SacI fragment of SoSPS1

including the coding region of the N-terminal part of SPS was ligated into pTrc99A

vector (Pharmacia) and the remaining coding region of SoSPS1 starting from XhoI up to

SphI (filled in) was inserted into the downstream of the N-terminal part. These steps

yielded pTrcSPS(Full) for the expression of full-length SPS under control of trc

promoter.

To produce SPS fused with a tag, the full coding region of SoSPS1 was

inserted into pTrcHisA with an N-terminal His6 tag (Life Technologies, Invitrogen).

The coding region was amplified by PCR using forward and backward primers

generating SbfI and EcoRI sites. The PCR amplified fragment was digested with SbfI

and EcoRI and inserted into PstI and EcoRI sites of the vector.

Expression and purification of SoSPS1 in Escherichia coli

The constructed plasmids were introduced into E. coli BL21 (DE3) competent

cells (Novagen) to express pTrc99A-SoSPS1, pTrcHisA-SoSPS1, and SoSPS1 mutants.

The transformed bacterial cells were grown in 50 ml of Luria Broth (LB) media

containing 50 µg/ml of ampicillin overnight at 30oC. The seed culture was inoculated

into a fermenter containing 8 L of LB media with 50 µg/ml of ampicillin and further

grown for 3-5 hr until an early log phase. Then, the culture temperature was shifted to

20oC, IPTG was added at a final concentration of 0.05 mM, and the culture was

continued overnight. The cells were harvested by centrifugation at 6,000 rpm for 6 min

at 4oC and stored at -20oC until use.

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The frozen bacterial cells were suspended in extraction buffer [50 mM

Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, and 1 mM PMSF], and

disrupted by sonication on ice. Cell homogenate was centrifuged at 15,000 rpm for 15

min at 4oC and the resulting supernatant was mixed gently with DE52 anion exchange

cellulose (Whatman). The mixture was filtered through Miracloth (Calbiochem) and the

filtrate was directly loaded onto a column of cOmplete His-Tag Purification Resin

(Roche) equilibrated with 50 mM Tris-HCl (pH 7.5) /150 mM NaCl. The column was

washed with the same buffer containing 30 mM imidazole and 10% glycerol (w/v) and

the resin binding proteins were eluted by increasing concentration of imidazole to 120

mM. The eluted sample from the His-tag resin column was concentrated and applied to

size exclusion chromatography on a Superdex 200 column (GE Healthcare Life

Sciences) and eluted with 50 mM Tris-HCl (pH 7.5) /150 mM NaCl.

Purification of the full length SPS without His tag was carried out by a

combination of ion-exchange, hydrophobic, and size exclusion chromatographies as

described in Fig. 2-1.

Partial purification of SPS from sugarcane and maize leaves

Frozen leaves were pulverized with liquid N2 and the proteins were extracted

in 3 volumes (v/w) of an extraction buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1

mM MgCl2, 1 mM EDTA, and 10% PVPP]. The homogenate was centrifuged at 14,000

rpm at 4oC for 15 min and the supernatant fluid was filtered through two layers

cheesecloth. Crude SPS was recovered by PEG-8000 fractionation between 6% and

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12% according to the method described previously (Torres, W. -K., et al 1987; Walker,

J. L., et al 1989). The SPS-containing precipitate was resuspended with 50 mM

Tris-HCl (pH 7.5) containing 150 mM NaCl and10% glycerol (v/v).

SDS-PAGE and Western blot analysis

Proteins in the total bacterial extract or in fractions obtained during

purification were separated by SDS polyacrylamide-gel (10%) electrophoresis (PAGE)

as described by Laemmli (1970). The gels were directly stained with Coommassie

Brilliant Blue or electroblotted onto Immobilon-P transfer membrane (Millipore) for

immunodetection. The polyclonal antibody was prepared against C-terminal 362 amino

acid residues of the coding region of SoSPS1. Proteins reacted with the antibody was

visualized with alkaline phosphatase-conjugated goat secondary antibodies (BioRad)

using the NBT/BCIP for color development. The quantification of protein bands stained

with Coommassie Brilliant Blue and visualized by the color development was

performed by densitometry using ImageJ software (http://imagej.nih.gov/ij/).

Assay of SPS activity

SPS activity was assayed as described previously (Huber, S. C., et al 1985)

with an appropriate modification. The assay mixture (50 µl) contained 50 mM

Hepes-NaOH (pH 7.5), 20 mM MgCl2, 20 mM F6P, and 20 mM UDP-G. The mixture

was incubated at 25-27oC for 10 min and reaction was stopped by an addition of 35 µl

of 1 M NaOH, followed by incubation at 95oC for 10 min to decompose unreacted F6P.

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To determine sucrose formed by the enzyme reaction, 125 µl of 0.1% (w/v) resolcinol

in 95% ethanol and 30% (w/v) HCl was added and heated at 85oC for 8 min. The

developed color of sucrose derivative was measured at absorbance 520 nm with using a

microtiter plate reader (SH-1000, Colona Electric).

Fig. 2-1 The pTrc99A-SPS was purified by a combination of several conventional

chromatographic steps. The purification procedure consisting of ion exchange

chromatography (DE52 and DEAE Toyo-pearl), hydrophobic chromatography (butyl

Toyo-pearl), gel filtration chromatography (superdex 200), and affinity chromatography

(blue-sepharose). Each purification step was monitored by enzymatic activity and the

histogram on the chromatography chart showed the activity of SPS.

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Result

Expression, purification and limited-proteolysis of SPS in E. coli

A full length cDNA of sugarcane SPS inserted into pTrc vector was introduced

into E. coli cells and its expression was checked by Western blot analysis of the total

bacterial cell extracts. As shown in Fig. 2-2A, two major bands with different mobility

on the SDS-PAGE gel were detected in the SPS gene transformed cells, but not in the

control cells; a larger band with around 120 kDa and a shorter one with 100 kDa were

designated as Form A and Form B, respectively. Form A was firstly appeared in the

bacterial cells at an early stage of growth, while Form B was increased in the cells of

prolonged cultivation. Enzyme activity of SPS was measured using the total extracts of

bacterial cells which accumulated the two forms with varying ratios (Fig. 2-2B). The

activity level was varied during a time course of the cultivation and the activity profile

was similar with the accumulation profile of Form B, suggesting that Form B might be

enzymatically more active than Form A.

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Fig. 2-2 Expression of SPS in E. coli cells. (A) Detection of SPS in E. coli transformed

with pTrcSPS(full) or the original vector pTrc99A (control vector). The bacterial cells

were grown for 0 to 24 hr and the total bacterial cell proteins were separated by

SDS-PAGE, followed by immunodetection with polyclonal antibodies against SPS

protein. Two major bands, Form A and B, were differentially accumulated in a

culture-time dependent manner. (B) Activity and accumulation levels of SPS. The total

extracts prepared from the comparable amounts of bacterial cells cultured for 4, 8, and

24 hr were subjected to activity assay and Western blotting. (C) Detection of SPS in E.

coli transformed with pTrcSPS(full) or pTrcHisSPS(full). The total bacterial cell

proteins were analyzed as in (A) by using polyclonal antibodies against SPS protein or

monoclonal antibody against the tag region. Sugarcane leaf extracts were also analyzed

in order to compare molecular sizes of authentic and recombinant SPSs.

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The gel mobility of the two forms of recombinant SPS was compared with SPS

prepared from sugarcane leaves and Form A was close in size with the authentic

enzyme (Fig. 2-2C). To examine whether Form B was a proteolytic product of Form A

by trimming the terminal region of SPS polypeptide, a 6-histidine-epitope-tag (His-tag)

was fused to the N-terminus of SPS and its expression profile was monitored by

immunoblotting using two different antibodies against SPS polypeptide and the epitope

tag. As shown in Fig. 2-2C, the His-tag SPS (HisFull) was also processed to be the two

forms, and Form A of HisFull retained the reactivity with the tag antibody as expected,

but Form B did not. This suggested that Form B was most likely a product with the

N-terminal region trimmed.

The two forms showed distinctive affinity with DEAE cellulose; Form A and

Form B were recovered in the unbound and bound fractions, respectively, and the

fraction containing Form B showed an activity higher than that containing Form A (Fig.

2-3). As shown in Fig. 2-1, Form B was successfully purified to a certain level of

homogeneity by a combination of conventional chromatographic procedures. No clear

date on the N-terminal amino acid sequence of Form B was obtained by automatic

Edman degradation procedure, suggesting that its N-terminus might be blocked. Form

A is hardly purified due to its non-binding nature to ion exchange resin (Fig. 2-3).

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Fig. 2-3 Partial purification to obtain Form A SPS. Recombinant SPS without His

tag (pTrc99A-SPS) was introduced into E. coli cells and two forms of SPS was

separated by DEAE cellulose. However, full length SPS (Form A) is hardly purified due

to its non-binding to ion exchange resin. The unbound protein containing Form A

fraction showed a lower activity than bound protein containing Form B fraction. The

bound protein showed rising of S6P formed by ten-fold than unbound protein.

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In vitro enzymatic regulation of wild-type and alanine substitution SoSPS1 mutant

Plant SPS was clearly activated by addition of G6P and deactivated by

addition of Pi. The effectors are also involved in the light modulation by reversible seryl

phosphorylation. G6P is known to be an allosteric effector in many plant SPSs. SPSs

from sugarcane and maize leaves are members of such allosteric enzymes. Maize SPS

has a high sequence homology with sugarcane SPS (Sugiharto, B., et al 1997). By using

SPS partially purified from maize leaves as a control of authentic enzyme, it was further

examined how allosteric characteristics were retained in Form B of recombinant

SoSPS1 and full-length SPS from maize SPS.

The present result shows the sigmoidal curve for maize SPS kinetic properties

and it indicated that maize SPS is activated on G6P activation, whereas, the Form B of

recombinant SoSPS1 has not been affected by G6P (Fig. 2-4).

Fig. 2-4 Kinetic properties of maize SPS and sugarcane recombinant SPS in the

presence of G6P

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Using the expression vector of the His-tag SPS, the deletion of N-terminal,

C-terminal, N- and C- terminal derivatives were designated as ∆N, ∆C, and ∆NC,

respectively. Their expression profiles were immunologically monitored using antibody

against the tag region. All genes encoding for the deletion region of SPSs were

expressed in the bacterial cells after overnight cultivation (Fig. 2-5). However, the

activity level showed the contrast result between deletion of N-terminal and C-terminal

regions. The activity of C-terminal deletion mutant was completely lost, while

N-terminal deletion mutant showed the retained SPS activity.

Fig. 2-5 The expression of N-terminal and C-terminal deletion of SPS

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Discussion

There are several papers reporting proteolytically cleaved of SPSs from plant

leaves in the course of purification to produce two fragments around 90-100 kDa and 30

kDa. Recombinant SPS expressed in the bacterial cells was also processed to a cleaved

form shorter by 20-30 kDa than the original size. All recombinant enzymes were shown

to be active, but it was not clearly described whether their activity was modulated by the

metabolic effectors or not. This is most probably because the full length and cleaved

enzymes could not be separately characterized. More extensive and detailed studies

were apparently necessary for understanding of structure-function relationship of plant

SPSs. In this study, it has been expressed sugarcane SPS in E. coli and observed that the

original SPS with a size of 120 kDa was trimmed down to be a shorter form with 100

kDa during bacterial cultivation. When His-tag was fused at the N-terminus of SPS, the

trimmed form had no tag-region (Fig. 2-2), indicating that the proteolytic cleavage

occurs at the N-terminal side.

It was also reported the expression of SPS from plant leaves in Escherichia

coli (Worrell, A. C., et al 1991; Sonnewald, U., et al 1993; Chen, W. L., et al 2007), but

resulting recombinant enzymes were not activated by G6P and Pi. The expressed

protein from E. coli did not show a clear property of the allosteric regulation and the

reason for that is not clearly understood yet (Chen, W. L., et al 2007). It suggested that

the loss of allostery properties due to the cleavage of N-terminal region. As some

bacterial SPSs lacking the N- and C-terminal regions of plant SPSs show no allosteric

regulation, possible allosteric properties of the truncated sugarcane enzyme are

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interesting.

To clarify the functional regulatory properties of sugarcane SPS, it was

expressed the mutant with N-terminal, C-terminal, both N- and C-terminal deletion. The

result showed that deletion of N-terminal SPS activity is retained. Conversely, deletion

of C-terminal region showed the loss of enzyme activity. It suggested that C-terminal

region of SPS has a crucial role in catalytic enzyme regulation and N-terminal deletion

is related to enzymatical properties and allostery regulation. Further detailed studies of

N-terminal region of sugarcane SPS will be undertaken to elucidate the mechanism of

SPS allostery regulation.

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Study 2.

The importance of N-terminal SoSPS1

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Introduction

Plant SPSs have a molecular mass around 120 kDa and consists of three

domains. The central region contains glycosyltransferase domain responsible for the

catalytic function of SPS (Castleden, C. K, et al 2004). The C-terminal region resembles

sucrose phosphate phosphatase and the N-terminal region has no clear similarity with

any other proteins. The SPSs from the photosynthetic cyanobacteria (Lunn, J. E., et al

1999) and nonphotosynthetic bacteria (Huynh, F., et al 2004) lack the N-terminal

domain or both terminal domains of plant SPS, respectively, and unlike plant SPS, their

enzyme activity is not allosterically regulated (Lunn, J. E., et al 2003a; Chua, T. K., et

al 2008). This indicates that the domain(s) characteristic of plant SPS is(are) contributed

to the regulatory properties and that, in particular, the unique N-terminal region is likely

to be involved in the regulation of enzyme activity.

In this study, cDNA of SoSPS1 was expressed in E. coli and insect cells and

recombinant enzymes with various lengths of the N-terminal deletion were produced.

According to the result of study 1, it suggested that the N-terminal region of sugarcane

SPS is not essential for the catalytic reaction itself, but plays a role in the allosteric

regulation.


Construction of N-terminal truncation SoSPS1 cDNA in E. coli

A series of mutant SPSs with the N-terminal truncation was also constructed

in a similar way of the his-tagged full-length enzyme. The truncations of the N-terminal

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region were generated by PCR using six different forward primers with SbfI site and the

backward primer containing EcoRI site as used for full-length SPS. The PCR product of

the N-terminal truncated forms designated as His∆N1, His∆N2, His∆N3, His∆N4,

His∆N5, and His∆N6 were subcloned into pGEM-T vector (Promega) that contains

3’-T overhangs at the insertion site and then cut with SbfI and EcoRI. The resulting

DNA fragments encoding the N-terminal truncated SPSs were inserted into PstI and

EcoRI sites of pTrcHisA. All the SPS genes thus constructed were verified by DNA

sequencing.

Expression and purification of N-terminal truncated of SoSPS1 in Escherichia coli

The constructed plasmids were introduced into E. coli BL21 (DE3) competent

cells (Novagen) to express the full length SPS and various N-terminal truncated forms.

The transformed bacterial cells were grown in 50 ml of Luria Broth (LB) media

containing 50 µg/ml of ampicillin overnight at 30oC. The seed culture was inoculated

into a fermenter containing 8 L of LB media with 50 µg/ml of ampicillin and further

grown for 3-5 hr until an early log phase. Then, the culture temperature was shifted to

20oC, isopropyl β-D-thiogalactopyranoside (IPTG) was added at a final concentration of

0.05 mM, and the culture was continued overnight. The cells were harvested by

centrifugation at 6,000 rpm for 6 min at 4oC and stored at -20oC until use.




27







mM. The eluted sample from the His-tag resin column was concentrated and applied to

size exclusion chromatography on a Superdex 200 column (GE Healthcare Life

Sciences) and eluted with 50 mM Tris-HCl (pH 7.5) /150 mM NaCl.

Expression of SoSPS1 in Sf9 insect cells and purification of the full-size SPS

SoSPS1 cDNA was PCR-amplified with primers shown in Fig. 3-1. A

5’-untranslated leader sequence of a lobster tropomyosin cDNA (L21) was included in

the forward primer for the enhancement of protein expression (Sano, K. I., et al 2002).

The PCR product was ligated into pGEM-T Easy vector (Promega), cut with NotI and

EcoRI, and then subcloned into the pFastBac1 (Thermo Fisher Scientific) vector for

baculovirus expression in Sf9 insect cells. A tobacco etch virus (TEV) protease

cleavage site, His8-myc-His8 tag, and EGFP were introduced at the carboxyl terminus of

SoSPS1.

The bacmid and baculovirus of SoSPS1 were generated using Bac-to-Bac

system (Thermo Fisher Scientific). Sf9 cells were grown to ~4.0 × 106 cells/ml in an

Erlenmeyer flask containing PSFM-J1 medium (Wako) and infected with P2 virus at

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28oC. After 24 hr post-infection, the cells were further incubated under low temperature

at 20oC for 48 hr. The cells were harvested by centrifugation at 4,000g for 5 min at 4oC,

resuspended in extraction buffer [50 mM Tris-HCl (pH7.5), 1 M NaCl, 20 mM

imidazole (pH7.5), 2 mM DTT, 2 mM benzamidine], and then disrupted by sonication.

The insoluble fraction was removed by centrifugation at 40,000g for 20 min at 4oC and

the supernatant was mixed gently with Ni-NTA Agarose (Qiagen) resin for 30 min at

4oC. The resin was washed thoroughly with wash buffer A [50 mM Tris-HCl (pH7.5),

0.3 M NaCl, 20 mM imidazole (pH7.5), 2 mM DTT]. SPS-EGFP was eluted with

elution buffer [50 mM Tris-HCl (pH7.5), 0.3 M NaCl, 250 mM imidazole (pH7.5), 2

mM DTT]. SPS-EGFP fraction was batch incubated with 2 ml of GFP-nanotrap resin

for 30 min at 4oC with gentle rotation (Rothbauer, U., et al 2008). The resin was washed

with wash buffer B [20 mM Tris-HCl (pH7.5), 0.5 M NaCl, 5 mM DTT]. SPS without

the tag was eluted from GFP-nanotrap resin by cleaving the linker between SPS and

His8-myc-His8-EGFP Tag with TEV protease for 4 hr at 4oC, leaving the tag bound to

the resin. The eluted SPS was concentrated and further purified by size-exclusion

chromatography on a column of Superdex 200 (GE Healthcare Life Sciences) in buffer

[20 mM Tris-HCl pH7.5, 0.5 M NaCl, 5 mM DTT].

Fig. 3-1 The primers that used to construct full-length SPS expressed in Sf9 insect cells

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SDS-PAGE and Western blot analysis

Proteins in the total bacterial extract or in fractions obtained during

purification were separated by SDS polyacrylamide-gel (10%) electrophoresis (PAGE)

as described by Laemmli (1970). The gels were directly stained with Coommassie

Brilliant Blue or electroblotted onto Immobilon-P transfer membrane (Millipore) for

immunodetection. The gels were directly stained with Coommassie Brilliant Blue or

electroblotted onto Immobilon-P transfer membrane (Millipore) for immunodetection

with the polyclonal antibodies against SPS or monoclonal antibody against Xpress™

epitope contained in the tag (Invitrogen). Proteins reacted with the antibody was

visualized with alkaline phosphatase-conjugated goat secondary antibodies (BioRad)

using the NBT/BCIP for color development. The quantification of protein bands stained

with Coommassie Brilliant Blue and visualized by the color development was

performed by densitometry using ImageJ software (http://imagej.nih.gov/ij/).

Assay of SPS activity

SPS activity was assayed as described previously (Huber, S. C., et al 1985)

with an appropriate modification. The assay mixture (50 µl) contained 50 mM

Hepes-NaOH (pH 7.5), 20 mM MgCl2, 20 mM F6P, and 20 mM UDP-G. The mixture

was incubated at 25-27oC for 10 min and reaction was stopped by an addition of 35 µl

of 1 M NaOH, followed by incubation at 95oC for 10 min to decompose unreacted F6P.

To determine sucrose formed by the enzyme reaction, 125 µl of 0.1% (w/v) resolcinol

in 95% ethanol and 30% (w/v) HCl was added and heated at 85oC for 8 min. The

30

developed color of sucrose derivative was measured at absorbance 520 nm with using a

microtiter plate reader (SH-1000, Colona Electric).

Result

Production of the N-terminal truncated forms of SPS

As the N-terminal truncation would give a significant effect on its enzymatic

nature as described above, a series of the N-terminal truncated forms of SPS with

different lengths deleted was considered to be useful for further investigation of an

effect of the N-terminal region, if any, on the enzyme activity. Using the expression

vector of the His-tag SPS as described above, the N-terminal truncated derivatives

designated as His∆N1 to His∆N6 were constructed (Fig. 3-2A). Their expression

profiles were immunologically monitored using two antibodies against SPS polypeptide

and the tag region (Fig. 3-2B). All genes encoding for the N-terminal truncated forms

were expressed in the bacterial cells and polypeptides with shorter sizes corresponding

to the length of truncation were found as a major band in bacterial cells grown for 4 hr,

which was reacted with both antibodies. After overnight cultivation, additional

polypeptide with the size equivalent with Form B appeared in all the truncated SPSs

except for His∆N6. These shorter polypeptides were not reacted with the tag specific

antibody, indicating that the limited proteolysis occurred irrespective of the length of

the N-terminal region. His∆N6 did not produce such shorter band and this was most

probably due to the lack of the proteolytic site, indicating that the cleavage site should

be within the regions between His∆N5 and His∆N6. It is noted that His∆N6 lost Ser162,

31

which was proposed to be one of phosphorylated serine residues involved in the

modulation of SPS activity (Sugiharto, B., et al 1997). Accumulation level of His∆N1

became lower than a limit of our immunological detection during the overnight growth.

Fig. 3-2 Expression of the N-terminal truncated forms of SPS in E. coli. (A) Sites of

the N-terminal truncation of SPS. SoSPS1 genes with deletion of the increasing length

of the N-terminal coding region were inserted into pTrcHis vector to construct fusion

with his-tag. The resulting truncated forms of SPS were designated as His∆N1 to

His∆N6. Ser162 marked with box is proposed to be a phosphorylation site (Sugiharto,

B., et al 1997). (B) E. coli cells transformed with the N-terminal truncated constructs

were grown for 4 hr or overnight. The total cell proteins from each transformant were

analyzed by Western blotting using antibodies against SPS polypeptide and the tag

region as in Fig. 1C. Major SPS bands with decreasing size corresponding to the length

of truncation were detected with both antibodies.

32

The N-terminal truncated forms of SPS were subjected to chromatography on a

Ni-resin column and representative data of His∆N5 is shown in Fig. 3-3A. This

purification step gave us an advantage to remove efficiently the limited proteolysis

product. Fractions containing His∆N5 were combined and further subjected to a size

exclusion chromatography. His∆N5 was eluted as a single major peak at a retention

volume corresponding to a molecular size of 370 to 380 kDa (Fig. 3-3C), indicative of

an oligomeric structure in a native state. His∆N4 and His∆N6 were also eluted in the

same profile with His∆N5 (data not shown). All truncated forms except for His∆N1

were thus successfully prepared by using the Ni-resin column as shown in Fig. 4A.

Purification of HisFull and His∆N1 was not successful, since they were hardly bound to

the Ni-resin column. The reason for this phenomenon was not clear, but it presumes that

the His-tag region might be masked within the whole structure of SPS molecule in a

manner not accessible with the resin. The presence of His-tag had been confirmed in an

SDS-denatured state by immunoblotting with the tag-specific antibody (Fig. 3-2B).

33

Fig. 3-3 Purification of the N-terminal truncated and full-length forms of SPS

expressed in E. coli and insect cells, respectively. (A) Chromatography of His∆N5 on

a column of Ni-NTA resin. (B) Sequential chromatography of Full (Sf9) on columns of

Ni-NTA resin and GFP nano-trap resin. (C) Size exclusion chromatography of His∆N5

and Full (Sf9). His∆N5 and Full (Sf9) obtained as described above were further purified

by size exclusion chromatography on a column of Superdex S-200.

Expression of SPS in insect cells

Preparation of the full length SPS was not successful in an E. coli recombinant

expression system. Then, baculovirus expression system using the Sf9 insect cell line

was applied. The C-terminus of SPS fused with His tag and EGFP was expressed in Sf9

34

cells and resulting expressed SPS was purified by successive chromatographies on

columns of Ni resin and GFP-nanotrap resin (Fig. 3-3B). The EGFP tag region was

removed by digestion with TEV protease and the full length SPS thus obtained,

Full(Sf9), was confirmed to have an molecular size equivalent with authentic SPS from

sugarcane leaves (data not shown). Full(Sf9) was subjected to a size exclusion

chromatography and eluted with an elution time comparable with that of His∆N5 (Fig.

3-3C).

Effect of the N-terminal truncation on enzyme activity

Activity of Form B and the truncation forms of His∆N2 to His∆N6, using at

least partially purified preparation (Fig. 3-4A), were measured in the presence of the

saturating concentrations of F6P and UDP-G, together with that of the full length SPS.

As shown in Fig. 3-4B, the truncated forms showed an increasing tendency of the

specific activity and this was the most remarkable for His∆N6 that showed an increase

in the activity higher by ten-fold than that of the full-length SPS. These results were in

good agreement with the above-mentioned observation that activity of SPS in the E. coli

cells became higher when accumulation level of Form B was increased over that of

Form A.

As the activity of plant SPS is known to be affected by G6P, assay were carried

out under increasing concentrations of two substrates in the presence or absence of G6P

(Fig. 3-5A and B). The activity of the full length SPS was significantly enhanced in the

presence of G6P and affinities for the two substrates were increased. The activity of the

35

N-terminal truncated forms was not significantly influenced by the addition of G6P

under all conditions tested in this experiment and this was a sharp contrast with the

results of full-length enzyme. It is noteworthy that affinities for UDP-G and F6P of the

N-terminal truncated forms were high even in the absence of G6P. These combine data

suggested that the N-terminal region of SPS, which is not proximal with the catalytic

domain in the primary structure, would give a significant generative influence on the

catalytic domain to lower enzyme activity and that thus, the deletions of this N-terminal

region was resulted in a disappearance of the suppressive effect.

It was further examined how allosteric characteristics were retained in the full

length and N-terminal truncated SPSs (His∆N3 and His∆N4). Under three different

concentrations of F6P, G6P-dependent activation of SPS activity was measured. As

shown in Fig. 3-6, the full length recombinant SPS was found to be activated by G6P in

a concentration dependent manner up to around 5 mM, and this tendency was

comparable with that of maize leaf SPS. Such clear allosteric property was not observed

in the N-terminal truncated enzymes.

36

Fig. 3-4 Comparison of activity of the N-terminal truncated and full-length forms

of SPS. (A) SDS-PAGE analysis of various forms of SPS used for activity measurement.

All the N-terminal truncated forms of SPS were prepared by His-tag affinity

chromatography as described in Fig. 3A. His∆N1 was not sufficiently obtained due to a

low expression in the bacterial cells (Fig. 3-2B). His∆N4, His∆N5, and His∆N6 were

further purified by size exclusion chromatography on a Superdex S-200 column. Full

(Sf9) was prepared as described in Fig. 3B. Amounts of sample loaded on a gel were

based on the activity unit to give an assay condition suitable for colorimetric

quantification of enzyme reaction. Protein bands were visualized by Coomassie Brilliant

Blue staining and quantification of various forms of SPS (protein bands marked by star)

were carried out by densitometry using imageJ software with an appropriate standard

protein. (B) Specific activity of the N-terminal truncated and full-length forms of SPS.

The enzyme activity was measured in the presence of saturated levels of two substrates,

10-20 mM UDP-G and 20 mM F6P. Specific activity was calculated with the

concentration of each enzyme determined as in (A).

37

Fig. 3-5 Kinetic properties of the N-terminal truncated and full-length forms of

SPS. (A) Activity of various forms of SPS was measured in the assay mixture

containing 20 mM UDP-G and increasing concentrations of F6P from 0 to 10 mM in

the presence or absence of 6 mM G6P. The amount of S6P formed for 10 min after an

addition of each enzyme was determined calorimetrically as described in materials and

methods. (B) Activity of various forms of SPS was measured in the assay mixture

containing 10 mM F6P and increasing concentrations of UDP-G from 0 to 20 mM in

the presence or absence of 6 mM G6P. Reaction rate was determined as in (A).

38

Fig. 3-6 An allosteric effect of G6P on the activity of recombinant SPSs and

authentic SPS from plant leaves. Activity of Full (Sf9), His∆N3, and His∆N4 was

measured in the assay mixture containing 20 mM UDP-G and 5, 10, or 20 mM F6P.

Effect of allosteric activator G6P was examined by the addition of increasing

concentrations from 0 to 8 mM. As a control for the allosteric activation of SPS by G6P,

a crude SPS fraction prepared from maize leaves was also assayed side by side.

39

Discussion

The activity of plant SPS, a key enzyme in sucrose biosynthesis, is modulated

by metabolic effectors, such as G6P and inorganic phosphate. To elucidate the

regulation of SPS activity, the enzyme needs to be prepared in an amount suitable for

kinetic analyses. It has obtained a series of mutants of SPS with the N-terminal

truncation of increasing lengths from 37 to 171 residues (Fig. 3-2A). Purification of

SPS with His-tag resin allowed us to separate the original and its trimmed forms of SPS.

As the full-length SPS was not successfully prepared in E. coli cells, we utilized insect

cells for preparation of recombinant SPS. The full-length enzyme was successfully

purified as a fusion with the C-terminal His-tag and EGFP, which could be cleaved off

during purification (Fig. 3-3B).

By kinetic analysis of the full length and truncated forms of sugarcane SPS

thus prepared, three important results were obtained: 1) the full length enzyme showed a

remarkable allosteric activation by G6P, while none of the truncated enzymes tested

here had such characteristics (Fig. 3-6), 2) specific activity of the full length and

truncated SPSs showed a tendency that longer truncation has higher activity (Fig. 3-4),

and 3) whether G6P was added or not, the truncated forms showed similar substrate

affinity with F6P and UDP-G, while the full length enzyme showed a higher substrate

affinity when G6P was present (Fig. 3-5A and 3-5B).

Present biochemical studies has clarified the functional roles of the

N-terminal region of sugarcane SPS. In conclusion, N-terminal region of sugarcane SPS

is crucial for the allosteric regulation, but no relationship between allosteric regulation

40

and specific activity. It was revealed by the truncated forms showed the

G6P-independent and that the increasing lengths of the deletions were resulted in an

enhancement of the catalytic efficiency. These results also indicated N-terminal region

may function like a suppressor domain for the enzyme activity. It would be necessary to

further analysis both the full length and the N-terminal truncated forms for elucidation

of the precise model of the allostery by crystal structure studies.

41

Study 3.

The structural studies of SoSPS1

42

Introduction

It has been reported the crystal structure of SPS from Halothermothrix orenii

and it reveals the mechanisms of substrate binding and glycosyl transfer in

nonphotosynthetic bacterium (Chua, T. K., et al 2008). However, the structural basis for

the regulatory function of plant SPS still remains unknown. Plant SPSs consist of three

domains, glucosyltransferase domain (GTD) (55 kDa), the C-terminal

phosphohydrolase domain (30 kDa) and the N-terminal domain (20 kDa). The

N-terminal domain has no apparent homology with known motifs (Cumino, A., et al

2002). Notably, cyanobacteria SPSs (Lunn, J. E., et al 1999) and H. orenii SPS

(HoSPS) (Chua, T. K., et al 2008), which lack the N-terminal domain of plant SPSs, are

not allosterically regulated. As noted above, modulation in plant SPS implies an

involvement of the N-terminal domain in the allosteric regulation.

According to the background of the research, it assumes that the combined

studies of functional and structural information from recombinant SoSPS1 may

represent an accurate model for reaction mechanism of SPS. Therefore, the objective of

research is characterization of recombinant SoSPS1 through crystal structure

determination.


Expression and purification of N-terminal truncated form of SoSPS1 cDNA in E. coli

Constructed plasmid of pTrcHisA-SPS was introduced into E. coli BL21

(DE3) competent cells (Novagen) to express His∆N5 and His∆N6 form of SPS. The

43

transformed bacterial cells were grown in 50 ml of Luria Broth (LB) media containing

50 µg/ml of ampicillin overnight at 30oC. The seed culture was inoculated into a

fermenter containing 8 L of LB media with 50 µg/ml of ampicillin and further grown

for 3-5 hr until an early log phase. Then, the culture temperature was shifted to 20oC,

isopropyl β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.05

mM, and the culture was continued overnight. The cells were harvested by

centrifugation at 6,000 rpm for 6 min at 4oC and stored at -20oC until use.










mM. The resulted sample from the His-tag resin column which contains SPS activity

was selected and applied for additional purification step onto Source-Q (GE Healthcare

Life Sciences) ion chromatography system. The eluted sample from Source-Q

chromatography system was concentrated and injected to size exclusion

chromatography on a Superdex 200 column (GE Healthcare Life Sciences) and eluted

with 50 mM Tris-HCl (pH 7.5) /150 mM NaCl.

44

Expression of SoSPS1 in Sf9 insect cells and purification of the full-size SPS

The bacmid and baculovirus of SoSPS1 were generated using Bac-to-Bac

system (Thermo Fisher Scientific). Sf9 cells were grown to ~4.0 × 106 cells/ml in an

Erlenmeyer flask containing PSFM-J1 medium (Wako) and infected with P2 virus at

28oC. After 24 hr post-infection, the cells were further incubated under low temperature

at 20oC for 48 hr. The cells were harvested by centrifugation at 4,000g for 5 min at 4oC,

resuspended in extraction buffer [50 mM Tris-HCl (pH7.5), 1 M NaCl, 20 mM

imidazole (pH7.5), 2 mM DTT, 2 mM benzamidine], and then disrupted by sonication.

The insoluble fraction was removed by centrifugation at 40,000g for 20 min at 4oC and

the supernatant was mixed gently with Ni-NTA Agarose (Qiagen) resin for 30 min at

4oC. The resin was washed thoroughly with wash buffer A [50 mM Tris-HCl (pH7.5),

0.3 M NaCl, 20 mM imidazole (pH7.5), 2 mM DTT]. SPS-EGFP was eluted with

elution buffer [50 mM Tris-HCl (pH7.5), 0.3 M NaCl, 250 mM imidazole (pH7.5), 2

mM DTT]. SPS-EGFP fraction was batch incubated with 2 ml of GFP-nanotrap resin

for 30 min at 4oC with gentle rotation (Rothbauer, U., et al 2008). The resin was washed

with wash buffer B [20 mM Tris-HCl (pH7.5), 0.5 M NaCl, 5 mM DTT]. SPS without

the tag was eluted from GFP-nanotrap resin by cleaving the linker between SPS and

His8-myc-His8-EGFP Tag with TEV protease for 4 hr at 4oC, leaving the tag bound to

the resin. The eluted SPS was concentrated and further purified by size-exclusion

chromatography on a column of Superdex 200 (GE Healthcare Life Sciences) in buffer

[20 mM Tris-HCl pH7.5, 0.5 M NaCl, 5 mM DTT].

45

Crystallization trial of N-terminal truncated form SPS

The final purified protein from size exclusion chromatography on a Superdex

200 column was concentrated to over than 6 mg/ml and conducted the crystallization

screening. The following 96-well with different temperatures and screening conditions

were tested for crystallization trial, such as SaltRx (Hampton Research) which screens

based on salt varying concentration and pH, PEGRx (Hampton Research) which is

using polymer reagents and pH in low to medium ionic strength, and Index (Hampton

Research) which is designed by specially formulated reagent zones to identify reagent

class and pH are effective to producing crystals. The 100nl of the protein sample was

aliquoted into the sitting drop well in 96-well plate using the Mosquito (TTP LabTech).

The remaining purified protein was further conducted Dynamic Light

Scattering (Malvern Instruments) for particle size characterization of protein and

Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS).

Result

Purification of the N-terminal truncated form expressed in E. coli and full-length SPS

expressed insect cells

The crude extracts of E. coli cells expressing N-terminal truncated forms,

His∆N5 and His∆N6, and crude extracts of insect cells expressing full-length SPS were

applied for various purification systems including ion exchange and the affinity

columns as described above.

The eluted samples of His∆N5 and Full (Sf9) were further purified by size

46

exclusion chromatography on a column of Superdex S-200. His∆N5 and Full (Sf9) were

eluted at comparable elution volumes of 10.4 ml and 10.5 ml, respectively. Calibration

of molecular size was carried out with marker proteins; thyrogloburin (669 kDa) at 9.1

ml, ferritin (440 kDa) at 10.2 ml, aldolase (158 kDa) at 12.1 ml, ovalbumin (44 kDa) at

14.8 ml, carbonic anhydrase (29 kDa) at 16.2 ml, and ribonuclease A (13.7 kDa) at 17.8

ml (Fig. 3-3C). These results suggested that the molecular size of native SPS from

N-terminal truncated form and full-length are similar about over than 120 kDa which

indicates the formation of oligomer.

Crystallization trial and particle size characterization of N-terminal truncated form SPS

The crystallization of His∆N6 was conducted with temperature up to 4oC and

20oC, however the result showed no crystal formed. In other hand, the molecular weight

of N-terminal truncated SPS has been analyzed using light scattering. Measurement of

molecular weight by SEC-MALS gives an indication of the oligomeric state (dimer).

Discussion

Our present data give a clear piece of evidence that the N-terminal region

composed of at least 171 residues of sugarcane SPS is crucial for its allosteric

sensitivity to G6P and that the N-terminal truncation as short as 37 residues leads the

mutant enzyme to be constitutively active with the allosteric sensitivity lost. The

N-terminal region of plant SPS was postulated to be intrinsic disordered by

bioinformatics analysis (Chua, T. K., et al 2008). It has been proposed that polypeptide

47

regions with intrinsic disorders play a role for modulation of allostery (Ferreon, A. C.,

et al 2013), such as optimizing high or low affinity binding and controlling the ability to

interact the protein surface (Hilser, V. J., et al 2007). Therefore, plant SPS might be one

of examples for such allosteric modulation.

The modular architecture of sucrose biosynthesis enzymes has recently been

suggested to represent oligomeric proteins, including SPS and sucrose synthase (SUS)

(Salerno, G. L., et al 2003). All SPSs and SUSs share 20-33% homology for the

sequences of GTD (Cumino, A., et al 2002) and the 3-D structures of GTDs from

Arabidopsis thaliana SUS (AtSUS) and bacterial SPS (HoSPS) were well superimposed

(Zheng, Y., et al 2011). Furthermore, AtSUS appears as a tetramer to generate the most

compact oligomer, which revealed the N-terminal half and C-terminal extensions

compromise the subunit interface (Zheng, Y., et al 2011). The similarity between GTDs

of SPSs and yeast glycogen synthase (Gsy2p) gave a possible mechanism for plant SPS

regulation (Chua, T. K., et al 2008). In eukaryotes, glycogen synthase is oligomeric and

the modulation of activity involves phosphorylation and G6P allostery (Pederson, B. A.,

et al 2000). The G6P binding to Gsy2p, leading a substantial conformation

reorganization at the subunit interface, increases the catalytic efficiency (Baskaran, S.,

et al 2010). By analogy, it could be hypothesized that the N-terminal domain of plant

SPS plays a direct role in allostery by inducing a structural perturbation upon binding to

G6P, which might include three-model states as an unphosphorylated state with

intermediate activity, phosphorylated state with reduced activity, and G6P-bound state

with high activity. Our present work has not given any information on phosphorylation

48

status of recombinant SPS except that loss of Ser162 in His∆N6, which is one of

phosphorylated serine residues (Sugiharto, B., et al 1997), has no significant effect on

SPS activity (Fig. 3-5) and further works will be necessary to draw more extensive story

of the allostery of plant SPS.

Based on these results, the modulation model of sugarcane SPS has been

proposed on Fig. 4-1. This model revealed that the oligomeric state has no correlation to

allosteric regulation at N-terminal region. The functional intactness of N-terminal

region in SPS has been noted like a suppressor domain for enzyme activity. In other

hand, binding of G6P at N-terminal region has induced the enhancement of UDP-G

substrate affinity binding and SPS becomes more active.

Fig. 4-1 A proposed model of sugarcane SPS for the functional intactness of

N-terminal region and allosteric regulation

49

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author(s) sawitri, widhi dyah citation issue date text ... study on structure and function of...

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